Field of the Invention
The present invention relates to a cell separation system, and in particular to a system for depleting certain blood components from normal blood, placental/umbilical cord blood, bone marrow, or stromal vascular fraction (SVF) cells once separated from adipose tissue.
Background of the Invention
Normal human blood generally comprises platelets (“PLTs”), plasma, red blood cells (“RBCs”), white blood cells (“WBCs”), and, in very small quantities, stem and progenitor cells (SPCs). On average (known to vary among individuals and, over time, within the same individual) RBCs make up approximately 99.9% of the number of an individual's total blood cells and account for approximately 45% of an individual's total blood volume. RBCs serve a vital function as the principal means of delivering oxygen to the body tissues. Nearly all of the remainder of an individual's blood volume is made up of plasma, a non-cell liquid component of blood accounting for approximately 55% of the total blood volume and in which all blood cells are suspended.
Thus, over 99% by volume of normal blood is made up of plasma and RBCs. The remaining approximately <0.6% by volume of normal blood consists of all other blood cell types and PLTs. PLTs are small, irregularly shaped anuclear cells that outnumber the WBCs by a factor of ˜10. PLTs play a fundamental role in wound care by stopping bleeding and releasing a multitude of growth factors that repair and regenerate damaged tissue.
The next most prevalent blood cells are WBCs, making up by number only about one tenth of one percent of the total cells in a typical blood sample. However, WBCs are critical to the body's immune system and defend the body against both infectious disease and foreign materials. The WBCs may be further divided into smaller subgroups. The largest such subgroup is granulocytes (GRNs), making up approximately 60% of all WBCs, and the other approximately 40% being mononuclear cells (MNCs). Throughout this application, the use of the term WBC may indicate a reference to exclusively GRNs, exclusively MNCs, or some combination of both.
MNCs can further be broken down into lymphocytes and monocytes, but may collectively be referred to as MNCs due to the presence in each cell of a single round nucleus. MNCs are critical elements of the immune system, comprising T cells, B cells and NK cells that migrate to sites of infection in body tissue and then divide and differentiate into macrophages and dendritic cells to elicit an immune response. Finally, the MNCs themselves can be further divided into even smaller subclasses—including extremely small quantities of multipotent hematopoietic (blood forming) stem and progenitor cells and mesenchymal (bone, fat, cartilage, muscle and skin forming) stem and progenitor cells, both critical to human health. Another source of MNCs are the stromal vascular fraction cells (SVFCs) that have been separated from adipocytes removed from individuals during liposuction.
Samples of normal blood, placental/umbilical cord blood or bone marrow are drawn in excess of 25 million times per year in the industrial world. Because the samples are generally taken either as a part of research into treatment of disease or for direct clinical treatment, the blood cells most often isolated are WBCs, followed by MNCs. MNCs include all the stem and progenitor cells, and approximately 40% of the critically important immune cells. Thus the cells most often in demand represent only a very small fraction of the cells drawn for a typical sample.
Thus if a relatively purified population of cells containing essentially all the stem and progenitor cells (SPCs) and depleted of substantially all the RBCs is desired, there is a need to separate the components of blood or bone marrow described above so as to isolate the WBCs or, if more purity is desired, the MNCs. This need for consistent, effective processes to separate these cell populations and harvest the target cells is especially pressing due to the increasing demand for SPCs for research, clinical trials, and point of care medical practices.
The interest in and research conducted on SPCs is staggering. As of November 2010 over 100,800 stem cell research articles have been published worldwide. There are currently at least 7,000 principle researchers focused on SPCs worldwide. In the United States alone there are some 300 stem cell research centers and approximately 10,000 individual labs. As a result of this extensive research has 199 clinical trials with cord blood stem cells, 34 clinical trials using adipose tissue, and 1,405 clinical trials using bone marrow stem cells are now underway according to clinicaltrials.gov, the NIH website.
Description of Related Art
Conventional methods of isolating and harvesting certain cell types from a whole blood or bone marrow aspirate sample generally involve centrifugation of the sample. During centrifugation, populations of cells tend to migrate to a relative position along the axis of lesser to greater acceleration according to their density, and concentrate in layers, displacing other higher and lower density cell types and plasma during the process.
FIG. 1 shows the density and average diameter of various cell types found in human blood. The physical differences between different cell types are important when the blood is centrifuged. When the blood is centrifuged the cells begin to move to new locations at velocities that are in accordance with many fluid dynamic factors, including Stokes Law. The fact that all cells retain a slight negative charge militates against direct cell-membrane-to-cell-membrane contact. In an environment comprising mainly plasma, with relatively few cells, the larger the cell, the more rapidly it travels. However as the concentration of cells rises, the effect of cell charge begins to substantially determine cell velocity.
However, in all cases, the denser the cell, the lower in the container (that is, further from the axis of centrifuge rotation) it will ultimately migrate to and settle. Thus, as shown in FIG. 2, the densest cells, RBCs (having a density between 1.08 and 1.12), will migrate to the bottom of the container being centrifuged. Within the RBC layer the nucleated red blood cells (which exist in both cord blood and bone marrow but not in normal blood) will be at the top of the red cell fraction. On top of the RBCs will be the GRNs (density 1.07-1.11), then, in order moving closer to the axis of rotation in the container, the lymphocytes (density 1.05-1.09), monocytes (density 1.045-1.0750) and the PLTs (1.03-1.065). It is known that the SPCs have a density closest to monocytes and lymphocytes and thus can be captured if those more numerous cells are captured. By taking advantage of the known strata that form under certain conditions, harvesting of one type of cell can be facilitated through the harvesting of only its strata. FIG. 2 also shows the relative frequency of the blood cell types in typical samples of normal blood, cord blood and bone marrow, and finally shows that there is some overlap in cell populations organized by density, as will be discussed below.
While creating the stratified cell layers generally requires nothing more than the application of high G forces over a set amount of time, precisely removing a specific layer of cells is problematic. To illustrate the rarity and small volume of certain cell populations in a given sample, FIGS. 3, 4, and 5 detail the respective average volumes of each cell population in normal blood (NB), cord blood (CB) and bone marrow (BM) after centrifugation and stratification.
FIGS. 3 and 4 illustrate that the vast majority of cells are RBCs, while FIG. 5 shows clearly that the volumes of non-RBC cellular blood components are so small that even with a 200 ml sample of cord blood the total volume of all GRNs (top line), MNCs (middle line) and PLTs (lower line) is about 1 mL. In cord blood fewer than 1 out of every 1,000 blood cells (approximately 0.08% of total cells) is an MNC. In a cord blood sample including 10,000 RBCs, one would expect to find 40-200 PLTs, 3-6 MNCs, and 5-10 granulocytes.
As explained, the vast majority of blood by both number of cells and by volume is made up of cells other than WBCs. Because of the scarcity of these WBCs and their residence within liquid solutions populated by enormous numbers of RBCs, current methods to isolate WBCs are (A) labor and time intensive, requiring excellent laboratory technique, (B) typically cannot be accomplished in a sterile environment, (C) typically have only between a 50-75% efficiency rate in capturing WBCs (a loss of 25% to 50% of the WBCs), and (D) involve processes that may adversely affect cell function. Given the typically small size of blood samples and the fact that SPCs are exceedingly rare in normal blood, there may be no SPCs at all in a typical harvest of the WBCs from normal blood and, although SPCs are more numerous in cord blood than in normal blood, they are still rare in cord blood.
To further illustrate how difficult it is to obtain WBCs from normal blood, a diagrammatic 50 ml test tube normally used in conventional manual blood component separation methods is shown in FIG. 6. These tubes are typically 28 mm in width. After centrifugation the separated blood components are the plasma (top) and the RBCs (bottom) and a nearly invisible thin layer called a “buffy coat” disposed in between (exaggerated in size in FIG. 6 for purposes of illustration). This “buffy coat” contains nearly all the WBCs, SPCs and PLTs.
Although several semi-automated systems for harvesting WBCs from whole blood are currently being marketed, their advantage in cell recovery efficiency relative to manual methods is not significant, and their market penetration is small. The prevalent current methods for isolating and capturing WBCs within a blood or bone marrow sample employ two manual processing technologies (A) the density gradient granules method and (B) the density gradient disk method. For diagnostic or research purposes, it is estimated that ninety-nine times out of a hundred when WBCs are isolated from blood or bone marrow they are isolated using these technologies. Both typically utilize cylindrical tubes for the process, and both rely on the careful manipulation of densities. For example, if the goal is to isolate MNCs, many thousands of tiny granules or a disk (of slightly smaller diameter than the tube inner diameter) with a density of approximately 1.08 are placed in the tube. This specific density value is chosen as it is equidistant between the median densities of GRNs and lymphocytes (see FIG. 2). In order to function correctly, both these technologies require that the blood sample first be diluted with an amount of buffer equal to 2 to 4 times the blood volume.
Referring first to the granule method, after the buffered blood sample is mixed with the granules in the test tube, centrifugation is initiated. During centrifugation, the density of the granules causes them to coalesce such that they divide the RBCs/GRNs from the MNCs/PLTs. FIG. 7 illustrates this process with Ficoll density gradient granules dividing the MNCs from the RBCs/GRNs.
The method of using density gradient disks is very similar and is illustrated in FIG. 8. Here, the disks of density 1.08 migrate under centrifugation to the interface between lymphocytes and GRN. However, most density gradient disks contain one feature not found in the granules above: they comprise a cavity between the upper and lower disks where it is expected the MNCs will settle, thus somewhat simplifying the step of harvesting of MNCs via a flexible tube that travels between the cavity and the top of the tube.
These manual methods for isolating and capturing MNCs from a blood sample require patience and excellent manual dexterity. These methods typically require 1½ to 2 hours to perform, and even with best practices, recovery of MNCs is often less than 60%. Thus the manual methods commonly employed for isolating and capturing MNCs within a blood sample are less than optimal in terms of precision and speed because of numerous limitations in this technology.
First, density gradient solutions achieve isolation of a population of WBCs from blood or bone marrow by relying on only one physical factor—density. Once the centrifugation begins, the density gradient medium moves to a position where it is buoyant in the cell solution and stops. Typically, this migration of the cell populations to their final positions occurs during an acceleration and duration that are both fixed, and thus rarely optimal for an individual blood sample.
Essentially the WBC or MNC harvesting efficiency of both density gradient technologies is limited by the need to aim at the center of the gap between the median density bell curves of the granulocytes and the lymphocytes (i.e., 1.08), as mentioned above with regard to FIG. 2. As FIG. 2 makes clear, this fixed density clearly does not exclude all the granulocytes or even all RBCs and does, in fact, discard some of the desired lymphocytes.
This simplistic approach also does not accommodate the fact that even in normally healthy people, there exists significant variation in the number and density of cells of each type and the sedimentation rates among samples may differ by as much as an order of magnitude. Further, if the patient has certain diseases the variation in the relative cell populations and the sedimentation rates of the cells can be much greater—up to two orders of magnitude. Consequently, these primitive WBC or MNC isolation technologies are rarely optimal for any specific sample of blood.
The best way to conceive of the severity of this limitation is to understand that the cells in a sample of blood, evenly distributed throughout the volume prior to centrifugation, begin a race to a new location when centrifugation begins. The efficiency of the WBC or MNC isolation technology depends upon how precisely all the WBCs wind up at the same strata at the end of the race—and how well the technician can extract the WBCs from this location with a pipette at normal 1G conditions where the cells will begin to remix with only the slightest motion of the container, or the slightest motion of the pipette tip.
Scientists have long studied the rate at which RBCs from normal blood migrate down a container under 1G conditions. This measurement is called the erythrocyte sedimentation rate, or ESR. Although the centrifugal acceleration used in conventional MNC isolation processes accelerate this rate of sedimentation, they do not change the percentage variation of the sedimentation rates of the different cell types. Further, as RBCs are more than 1000 times as numerous as WBCs and 2000 times as numerous as MNCs, and all the cells maintain a slight negative charge, it is the RBC migration that most effects isolating the WBC populations.
The ESR (measured in millimeters per hour—mm/hour) in adults of various age are shown in FIG. 9. In children, normal values of ESR have been found to be 1 to 2 mm/hour at birth, rising to 4 mm/hour 8 days after delivery, and then to 17 mm/hour by day 14 (a change of more than an order of magnitude in less than two weeks). The ESR is so variable that it is used to diagnose malignant diseases, such as multiple-myeloma, various auto-immune diseases such as rheumatoid arthritis, and chronic kidney diseases wherein the ESR may exceed 100 mm/hour, five times that of a normal adult.
Further, it is noted that WBCs at the bottom of a container can only move upward to join those descending from the top of the container as a result of being buoyed up on the ascending plasma displaced by the descending RBCs. Note that the very small number of WBCs in a solution must negotiate their path upwards against the flow of RBCs, a thousand times more numerous, moving in the opposite direction through the same vertical channel. Further, as the acceleration and duration of the centrifuge is programmed at the start of the run, a duration that is satisfactory to relocate all the cells within a specific sample may be insufficient for many other blood samples in that most of the RBCs may not have arrived at the bottom of the tube and thus many of the target WBCs may not have been buoyed up to the “buffy-coat” strata by the ascending plasma. As this process takes place in a closed centrifuge within a rapidly spinning rotor, the operator is unable to observe the actual motion of the cells.
There is thus a need for a system, which optically tracks, in real time, the migration of cell populations within each individual blood sample during centrifugation. Such a system would allow each individual blood sample to be custom processed according to the specific size and density of that blood sample's constituent cell populations. This improved solution should also greatly increase the harvesting efficiency of target WBC or MNC cell populations.
A further drawback to density gradient mediums is that they require buffers, which occupy most of the volume of a given harvest tube, minimizing the volume of blood from which WBCs are to be harvested. A buffer often occupies 70% to 90% of the 50 ml volume of a typical harvest tube, leaving space for only 5 to 15 ml of blood. Consequently, a technician who needs to harvest WBCs from 100 ml of blood must employ 7 to 20 test tubes—further increasing the labor required to accomplish the goal. Additionally, in order to achieve adequate purity from contaminants in the final WBC population, the granular density gradient mediums and the buffers will need to be washed out, inevitably causing a further loss of target cells.
There is thus a need for a means for depleting undesired cells from a blood or bone marrow sample, which does not require voluminous density gradient mediums or buffers. The means may optionally allow the harvest of WBCs from larger volume samples, further increasing the number of constituent cells that may be recovered for diagnostic or clinical use.
A third drawback to the density gradient based blood separation methods described above is that the parallel vertical walls of a density gradient harvest tube do not assist the WBCs rising during centrifugation to lie atop the RBCs. The density gradient harvest test tubes in conventional systems have vertical parallel walls meaning that during centrifugation all the cells either fall or rise vertically along the axis of the tube. As described above, each ascending WBC must negotiate thousands of RBCs moving in the opposite direction. The harvest test tube's parallel vertical walls provide no lateral motion to descending RBCs and ascending WBCs that could assist the rise of the WBCs during centrifugation. As a result a significant portion of the WBCs that began the spin cycle in the bottom of the tube may not rise to the harvest “buffy coat” layer during the chosen centrifugation regimen.
There is thus a need to overcome the entrapment of WBCs at the bottom of the test tube through utilization of a funnel-shaped harvest chamber that radically narrows at the bottom, such that most of the descending RBCs are forced to the center, enhancing eddy currents led by the lightest of the RBCs ascending to the top of the RBC volume. In turn these eddy currents assist the ascension of the much less numerous but more buoyant WBCs.
A fourth drawback to the density gradient based separation methods is the constant large cross sectional area of the density gradient harvest test tube at the location where the MNCs are harvested manually at 1 G.
Because the walls of a standard 50 ml density gradient harvest tube are a fixed ˜28 mm apart, the very small volume of MNCs from a 10 ml peripheral blood sample (˜0.028 ml) are spread across the entire cross sectional area of the tube (˜615 mm2) in a thin layer (˜0.023 mm) which is nearly invisible. Because of this broad cross sectional area and the resulting thin layer of MNCs, the stratifying effects of the density differences between cell populations are miniscule. Consequently, harvesting the MNCs at 1 G requires a highly trained technician to slowly and carefully insert a pipette tip into this very thin layer of cells that floats between the density gradient (below) and plasma (above) and then gently apply a suction to draw the MNCs up into the pipette. However, the very small density variations between cell populations, when not magnified by substantial centrifugal forces, and the large cross sectional area of the tube conspire to keep the MNCs/PLTs essentially all in the same thin vertical layer. Consequently, no amount of care during this manual suction process prevents the roiling of the MNC/PLT layer and the density gradient media so a loss of MNCs and substantial contamination of the cells by the density gradient granules ensues. It is thus not uncommon to lose ˜25-40% of the MNCs during this procedure.
There is thus a need to avoid losses during the harvest of MNCs by depleting RBCs and most of the GRNs through the narrow cross sectional area of a funnel exit while substantial centrifugation maintains the integrity and purity of the cell strata and elongates them vertically as they descend down the tapered funnel.
A fifth drawback to conventional density gradient granule based systems is due to the direct contact between the density gradient granules and the cells. The extensive direct contact between these granules and the cells to be harvested has been reported to damage the cell function due to a form of toxicity. For example, Yuhan Chang, et al recently reported in “The Efficiency of Percoll and Ficoll Density Gradient Media in the Isolation of Marrow Derived Human Mesenchymal Stem Cells with Osteogenic Potential” (Chang Gung Med J 2009; 32:264-75) that when cytoxicity tests were run on CFU-Fs (passage one) by culturing with a mixture of control medium and Percoll or Ficoll in serial dilutions to assess the growth-inhibitory or cytotoxic effects of these two gradient media, the CFU-Fs exhibited greater cell death as the ratio of gradient medium increased in both groups.
There is thus a need to provide for the depletion of RBCs, or RBCs and GRNs, or RBCs, GRNs and PLTs, or RBCs, GRNs and MNCs, without requiring the addition of density gradient granules or any other foreign matter that may alter or damage cell function.
A sixth drawback of conventional blood separation methods, with or without density gradient granules, is the probability that significant numbers of RBCs may remain in the final product due to the variability in technician competence and the ease of inadvertently remixing the cells at 1 G. Several recent studies have highlighted the importance of minimizing RBC contamination because such contamination decreases the efficacy of medical treatments using these MNCs.
Examples of these mal effects of RBC contamination abound, for example see “Red Blood Cell Contamination of the Final Cell Product Impairs the Efficacy of Autologous Bone Marrow Mononuclear Cell Therapy,” Assmus et al., Journal of the American College of Cardiology, 55.13, 2010, wherein it is disclosed that contaminating RBCs affect the functionality of isolated BMCs and determine the extent of left ventricular ejection fraction recovery after intracoronary BMC infusion in patients with acute myocardial infarction. See also, “Packed Red Blood Cells Suppress T-Cell Proliferation Through a Process Involving Cell-Cell Contact,” Bernard et al., The Journal of Trauma, Injury, Infection, and Critical Care, 69.2, August 2010, wherein it is disclosed that stored RBCs exert a potent inhibitory effect on T-cell proliferation, and it is likely that similar suppression of T-cell proliferation could occur in vivo after blood transfusion and may be a major contributor to transfusion related immunomodulation.
There is thus a need to provide for the greater and more predictable depletion of RBCs, GRNs and, possibly, PLTs from a collected sample of normal or cord blood, bone marrow or SVF cells separated from adipose tissue in order to recover a more purified solution containing SPCs.
The few commercially available systems that have automated this cell separation and depletion process (such as the Hemonetics V50, the Cobe Spectra, the Sepax System by Biosafe SA of Switzerland, and the Thermogenesis AXP by Thermogenesis Corp. of California) also have substantial drawbacks and have not achieved improved recoveries of purified WBCs relative to the conventional manual means. An additional drawback is that these commercially available automated systems require expensive capital equipment in order to operate. These automated devices cost tens of thousands of dollars and occupy substantial laboratory space if significant production of units of purified WBCs are required—such as with cord blood stem cell banks that may process 40 to 200 units per day. FIG. 10 illustrates the costs of processing four units of blood with two prior art systems and with the current system.
A second drawback of the currently commercially available automated systems is that they require complicated, expensive, difficult to manufacture single use disposable bag sets linked together with substantial tubing to process the cells, as shown in FIG. 11. These bag sets take approximately five minutes to correctly load into their dedicated devices and to ready the system to process the blood or bone marrow. These prior art bag sets are complex and costly to manufacture. As shown in FIG. 11 these prior art bag sets require more than 20 individually formed glue joints.
There is thus a need for a simpler, less expensive, faster and easier to use automated system that is also able to achieve higher recoveries of WBCs with less contamination by RBCs. There is further a need for a system that employs a simple, inexpensive to manufacture single-use disposable processing container, which does not require multiple bags and complex connecting tubing.
FIG. 12 illustrates the simplicity of the current invention in that it provides an all-in-one cylindrical cartridge in which all cell processing occurs and in which all components related to cell stratification and depletion are disposed. In as few as one or two seconds this cartridge may lock onto the top of a dedicated cylindrical control module and be ready for insertion into a centrifuge cup. The control module contains optical and gravitational sensing means as well as means for controlling the activity in the cartridge.
This all in one cartridge benefits from the manufacturing precision of injection molding and is much simpler and labor efficient to construct than conventional processing disposables for prior art automated systems typically comprising multiple bag sets and complicated connecting tubing connected thereto.
It is thus a first objective of the present invention to optically track the migration of the cell populations for each individual blood sample and to then deplete certain cell types by diverting them into a secondary and separate compartment within the same cartridge during centrifugation.
It is a second objective of the present invention to provide for the selective depletion of substantially all unwanted cell types while not requiring volume consuming density gradient mediums or buffers.
It is a third objective of the present invention to provide a rigid funnel shaped harvest chamber that is substantially narrower at its bottom portion such that descending RBCs are forced to the center of the funnel, thereby enhancing vertical eddy currents led by the lightest of the RBCs ascending to the top of the red cell volume which assists the ascension of the much less numerous but more buoyant WBCs to the initial WBC stratification and concentration level.
It is a fourth objective of the present invention to provide an all-in-one cartridge in which all cell processing occurs and in which all components related to cell stratification and depletion are located at the completion of the centrifugation. The cartridge may be easily, quickly and removably locked to a control module that, under centrifugation, relies on its own strength for support rather than a support structure in which it is nested. This cartridge preferably comprises at least three rigid compartments: (1) The RBC compartment into which the bulk RBCs and, at operator discretion, unwanted GRNs are directed; (2) the stem cell (SC) compartment into which the targeted WBCs are directed which may include, at operator discretion, GRNs, lymphocytes, monocytes, SPCs and/or platelets; and (3) the harvest funnel which initially contains the entire sample of blood or bone marrow and retains, after processing, any excess plasma.
It is a fifth objective of the present invention to create a layer of WBCs within a funnel that, when urged downwards by centrifugal force, encounter a portion of the funnel of decreasing diameter, thereby causing said WBC layer to increase in vertical thickness.
It is a sixth objective of the present invention to provide a means for stratifying a blood sample into RBCs, GRNs, MNCs, PLTs and plasma, and for precisely opening and closing certain valves at the interface between certain cell layers.
It is a seventh objective of the present invention to provide a means of harvesting a higher percentage of the WBCs while simultaneously depleting a higher percentage of RBCs than is obtainable using conventional manual or automated systems and without the requirement of RBC sedimentation agents such as hetastarch.
It is an eighth objective of the present invention to provide the above seven objectives at a reduced cost as compared to conventional manual and automated systems currently in place.
These and other objectives, advantages, features, and aspects of the present invention will become apparent as the following description proceeds. To the accomplishment of the foregoing and related ends, the invention, then, comprises the features hereinafter more fully described and particularly pointed out in the claims, the following description and the annexed drawings setting forth in detail certain illustrative embodiments of the invention, these being indicative, however, of but several of the various ways in which the principles of the invention may be employed